Graphical AbstractHighlights d ORP2 is a unique transporter of both cholesterol and PI(4,5)P 2 d ORP2 forms a tetramer to efficiently transfer PI(4,5)P 2 d ORP2 controls the levels of both cholesterol and PI(4,5)P 2 on the plasma membrane d ORP2 associates with the plasma membrane via binding to PI(4,5)P 2 In BriefCholesterol and PI(4,5)P 2 are two key lipid components of the plasma membrane. Wang et al. identify ORP2 as a unique transporter of both cholesterol and PI(4,5) P 2 . ORP2 regulates the levels of both cholesterol and PI(4,5)P 2 on the plasma membrane, and ORP2 forms a tetramer to efficiently transfer PI(4,5)P 2 . SUMMARYCholesterol is highly enriched at the plasma membrane (PM), and lipid transfer proteins may deliver cholesterol to the PM in a nonvesicular manner.Here, through a mini-screen, we identified the oxysterol binding protein (OSBP)-related protein 2 (ORP2) as a novel mediator of selective cholesterol delivery to the PM. Interestingly, ORP2-mediated enrichment of PM cholesterol was coupled with the removal of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P 2 ) from the PM. ORP2 overexpression or deficiency impacted the levels of PM cholesterol and PI(4,5)P 2 , and ORP2 efficiently transferred both cholesterol and PI(4,5)P 2 in vitro. We determined the structure of ORP2 in complex with PI(4,5)P 2 at 2.7 Å resolution. ORP2 formed a stable tetramer in the presence of PI(4,5)P 2 , and tetramerization was required for ORP2 to transfer PI(4,5)P 2 . Our results identify a novel pathway for cholesterol delivery to the PM and establish ORP2 as a key regulator of both cholesterol and PI(4,5)P 2 of the PM.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.

Background: Caveolin-1 (Cav1) requires the caveolin scaffolding domain for caveola formation. Results: The Cav1 scaffolding domain and oligomerization domain are tightly juxtaposed to the membrane in caveolae. Conclusion: Concerted membrane association of the oligomerization, scaffolding, and intramembrane domains are critical for caveola biogenesis and membrane deformation. Significance: Understanding the membrane association of Cav1 is critical for dissecting how the protein regulates caveola formation and achieves regulation over cellular signaling.

Highlights d A strategy for improving the ADCC potential of therapeutic antibodies is presented d Temporary inhibition of endocytosis increases tumor cell antigen presentation d Prochlorperazine could be repurposed to enhance the efficacy of anti-tumor mAbs d Potential to reduce heterogeneity in tumor cell responses to many IgG1 antibodies